Sample Testing Systems And Methods

ABSTRACT

A testing arrangement and testing method using a manipulator assembly configured to manipulate an apertured surface to and from a sample area and a cleaning area and a controller configured to control the manipulator assembly, wherein the controller includes a sample module configured to control the apertured surface through the sample area, which is configured to contain biological sample targets, and wherein the control system enables re-use of the apertured surface with at least two different biological sample targets.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/232,770, filed 9 Aug. 2016, which claims benefit under 35 USC §119(e) of U.S. Provisional Patent Application 62/202,858 filed 9 Aug.2015, each of which is incorporated herein by reference in its entiretyas if set forth herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

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THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

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SEQUENCE LISTING

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STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINTINVENTOR Not Applicable BACKGROUND OF THE DISCLOSURE 1. Field of theInvention

The present invention relates to sample testing systems and methods, andmore particularly to biological sample testing systems and methods thatenable re-use of the same testing tip over two or more testingprotocols.

2. Description of Related Art

Patch-clamp recording is a gold-standard single-cell electrophysiologytechnique that has been widely used to discover foundational biophysicalproperties of excitable cells. In neuroscience, the superior sensitivityand resolution of patch-clamp recording has made it an indispensabletool for discovering the tenants of ion channel activity, synapticintegration, plasticity and network connectivity in a variety ofexperimental preparations from cultured cells to living tissue.

Many pathological conditions such as migraine, hypertension andhypersomnia arise from dysfunctions in cell membrane-bound ion channels.Patch clamp recording is the highest resolution tool available tomeasure the responses of these channels to drug treatments. Patch-clamprecording provides an unprecedented ability to measure extremely small(on the order of 10⁻¹² Amperes) electrical currents arising as a resultof single or multiple ion channels trafficking charged ions across thecell membrane.

There is a growing demand for large-scale single-cell measurements ofelectrical and molecular activity; for example, for drug screening, celltype characterization, and biophysical analysis. Patch-clamp iswell-suited for such studies because it can sample electrical,morphological and genetic properties of single cells and evensub-cellular compartments; however, the technique is not readilyscalable.

Patch-clamp recording involves pressing a glass micro-pipette (namelythe aperture) against a cell membrane to achieve a high-resistance (>1GΩ) seal, or “gigaseal”, with the cell membrane. These gigaseals onlyform when using clean micro-pipettes, necessitating an operator toreplace the contaminated pipette with a fresh one between every contactwith a cell membrane. This is a major barrier to facilitatinghigh-throughput drug screening.

In this “conventional” approach to patch-clamp recording, a glasspatch-clamp electrode (a hollow glass capillary pulled to a fine ˜1 μmdiameter aperture at the distal end) is gently pressed against themembrane of a cell, typically under microscope guidance. A briefapplication of suction to the pipette forces the cell membrane to fullyocclude the aperture at the distal end of the electrode. This results inthe formation of an intimate physical connection between the cellmembrane and the pipette aperture, termed a “gigaseal” because theresistance between the membrane-occluded pipette aperture and anextracellular ground electrode is equal to or greater than 1 gigaohm.

In both conventional and planar patch-clamp systems, to achieve asuccessful recording, the patch-clamp aperture must have a clean tip toform a high-resistance (≥1 GΩ) junction with the cell membrane. In hisNobel Prize lecture, Edwin Neher remarked that “a gigaseal could beobtained reproducibly when suction was combined with some simplemeasures to provide for clean surfaces, such as using a fresh pipettefor each approach and using filtered solutions”. However, the need for afresh pipette for each approach or a new planar chip is a majorlimitation to automation and throughput. In conventional systems, theubiquitous practice of exchanging pipettes requires dexterity from theexperimenter, especially when performing simultaneous patch-clamprecordings with multiple pipettes. In planar patch-clamp systems, theneed to replace chips after every recording comes at a significant costto the user.

Further, some promising studies are impractical at large scales becausepipettes are not easily replaceable, for instance if they are coated,custom-manufactured, or filled with precious molecules such as syntheticpeptides, novel therapeutics, human patient samples or nucleic acidconstructs.

As noted above, and confirmed by Dr. Neher, it is widely accepted thatcleanliness of the electrode is paramount for the formation of agigaseal and that once an electrode has come in contact with a cellmembrane, the electrode cannot be used to form a subsequent gigasealwith another cell due to adherent cellular debris. This necessitatesusing fresh electrodes for every patch-clamp trial in both conventionaland planar patch systems.

It is thus an intention of the present invention to provide a simple,fast, and automated method for cleaning glass pipette electrodes thatenables their immediate re-use. By enabling a single pipette to carryout multiple patch-clamp recordings, full automation of the techniquecould be achieved which would increase throughput dramatically.

BRIEF SUMMARY OF THE INVENTION

In an exemplary embodiment of the present invention, a control systemcomprises a manipulator assembly configured to manipulate an aperturedsurface to and from a sample area and a cleaning area and a controllerconfigured to control the manipulator assembly, wherein the controllercomprises a sample module configured to control the apertured surfacethrough the sample area, which is configured to contain biologicalsample targets, and wherein the control system enables re-use of theapertured surface with at least two different biological sample targets.

The controller can further comprise a cleaning module configured tocontrol the apertured surface through the cleaning area configured tocontain cleaning solution, the cleaning area fluidically separate fromthe sample area, and a testing module configured to control theapertured surface into contact with a biological sample target of thebiological sample targets to test for at least one characteristic of thebiological sample target.

The cleaning module can be configured to control an automated pressureassembly in communication with the apertured surface, wherein thecleaning module can be further configured to control the application, bythe automated pressure assembly, from negative to positive pressuresrelative to atmosphere to draw toward and expel from the aperturedsurface at least a portion of cleaning solution in the cleaning area,and wherein the cleaning module can be further configured to control theamount of cleaning solution drawn into the apertured surface so itreaches a height of a testing implement having the apertured surface,the height determined to sufficiently clean remnants of the testedsample target located at and beyond the apertured surface.

In another exemplary embodiment of the present invention, a biologicalsample testing system comprises the control system described above, theapertured surface, and a re-use assembly comprising the sample area andthe cleaning area configured to contain cleaning solution, the cleaningarea fluidically separate from the sample area, wherein the controlleris further configured to manipulate the apertured surface into contactwith a biological sample target of the biological sample targets to testfor at least one characteristic of the biological sample target, andmanipulate the apertured surface to the cleaning area, wherein remnantsof the tested biological sample target are cleaned from the aperturedsurface.

In another exemplary embodiment of the present invention, a biologicalsample testing system comprises a testing implement with a testing tiphaving an aperture, a manipulator assembly configured to manipulate thetesting implement, and a re-use assembly comprising a sample areaconfigured to contain biological sample targets, a cleaning areaconfigured to contain cleaning solution, the cleaning area fluidicallyseparate from the sample area, wherein the manipulator assembly isconfigured to manipulate the aperture of the testing implement intocontact with a biological sample target of the biological sample targetsto test for at least one characteristic of the biological sample target,and manipulate the testing implement to the cleaning area, whereinremnants of the tested biological sample target are cleaned from thetesting implement, wherein the re-use assembly enables re-use of thetesting tip of the testing implement with at least two differentbiological sample targets.

At least one characteristic of the tested biological sample target canbe selected from the group consisting of an electrical activity, amolecular activity, a drug screening property, biological sample targettype, a biophysical property, a morphological property and a geneticproperty.

The biological sample testing system can further comprise a controllerconfigured to control the manipulator assembly and the re-use assembly.

The manipulator assembly can be configured to manipulate the testingimplement to the cleaning area whether or not the test for at least onecharacteristic of the biological sample target is successful.

The biological sample testing system can further comprise an automatedpressure assembly in communication with the testing tip of the testingimplement, wherein the automated pressure assembly is configured toapply from negative to positive pressures relative to atmosphere to drawtoward and expel from the aperture at least a portion of cleaningsolution in the cleaning area, and wherein cleaning solution is drawn aheight into the testing implement sufficient to clean remnants of thetested sample target located at and beyond the aperture.

The biological sample testing system can further comprise at least asecond testing implement with a testing tip having an aperture.

The sample area can comprise two or more discrete sample unitsfluidically separate from one another.

The cleaning area can comprise two or more discrete cleaning unitsfluidically separate from one another.

The controller can be a software control module.

The manipulator assembly can comprises an automated micro- ornano-positioner, wherein the controller can be configured toautomatically control manipulation of the aperture of the testingimplement into contact with a first biological sample target of thebiological sample targets, after the test, manipulation of the testingimplement to the cleaning area, after cleaning, manipulation of thetesting implement to the sample area, manipulation of the aperture ofthe testing implement into contact with a second biological sampletarget of the biological sample targets, the second biological sampletarget different than the first biological sample target, and after thetest, manipulation of the testing implement to the cleaning area.

The controller can further comprise hardware embedded with the softwarecontrol module.

The test of the first biological sample target and the test of thesecond biological sample target can be separately selected from thegroup consisting of successfully testing at least one characteristic andunsuccessfully testing at least one characteristic.

In another exemplary embodiment of the present invention, a method ofbiological sample testing comprises manipulating an apertured surface toand from a sample area and a cleaning area, controlling the aperturedsurface through the sample area, which is configured to containbiological sample targets, and re-using the apertured surface with atleast two different biological sample targets.

Controlling the apertured surface through the sample area can comprisecontacting the apertured surface with a first biological sample targetof the biological sample targets, and testing for at least onecharacteristic of the first biological sample target, and whereinre-using the apertured surface can comprise cleaning remnants of thetested biological sample target from the apertured surface, andcontacting the apertured surface with a second biological sample targetof the biological sample targets.

At least one characteristic of the tested biological sample targets canbe selected from the group consisting of an electrical activity, amolecular activity, a drug screening property, biological sample targettype, a biophysical property, a morphological property and a geneticproperty.

Cleaning remnants of the tested biological sample target from theapertured surface can comprise applying from negative to positivepressures relative to atmosphere to draw toward and expel from theapertured surface at least a portion of cleaning solution in thecleaning area, and drawing a volume of cleaning solution into aperturedsurface sufficient to clean remnants of the tested sample target locatedat and beyond the apertured surface.

In another exemplary embodiment of the present invention, a method ofstimulating and/or measuring one or more physical characteristics of atest target located in a test area comprises moving an apertured surface(for example, a micro-pipette/-electrode/-probe tip) with amicro-/nano-positioner such as micromanipulator into physical contactwith the surface of the target surface, forming a quality contact andinterface between the apertured surface and the test target surface inorder to test at least one characteristic of the test target selectedfrom the group consisting of an electrical activity, a molecularactivity, a drug screening property, target type, a biophysicalproperty, a morphological property and a genetic property, after testingor a failed attempt at the testing, preparing the apertured surface andsubstantially immediate inner surface(s) of the tip behind the aperturedsurface by moving the tip with the apertured surface from the test areato a cleaning area, cleaning the apertured surface and substantiallyimmediate inner surface of the tip with cleaning solution in thecleaning area that is separate from the test area via at least onealternating positive and negative pressure cycle to draw toward andexpel from the apertured surface at least a portion of cleaning solutioninside the tip, moving the tip from the cleaning area after cleaning tothe test area, and forming a quality contact interface with another testtarget, wherein preparing the apertured surface and substantiallyimmediate inner surface(s) of the tip behind the aperture enables re-useof the same micro-pipette/-electrode/-probe with at least twoconsecutive, but different, test targets.

The testing can involve recording/measuring from the test target, aswell as stimulating it during such measurements (e.g., in electricalrecordings, the invention can include exposing the test target to acontrolled voltage or current while measuring its response to it.

Preferably, steps are automated, namely at least preparing themicro-pipette/-electrode/-probe tip by the agency of an automatedprocess.

Preparing the apertured surface and substantially immediate innersurface(s) of the tip behind the apertured surface can further compriserinsing it after cleaning it, by moving the tip to the test arealocation away from a next test target or a separate rinse area withsolution that is not harmful to the test target, and applying at leastone positive or negative pressure pulse.

Motorized/automated micro-positioners/manipulators and an automatedpressure controller can automate the process, andprocessors/memory/software can be used to implement the automation.

In an exemplary embodiment of the present invention, an operator of thesystem does not touch anything during testing, cleaning and moving to anext test target/cell, which manual interference can cause vibrationsand other mechanical instabilities, which often lead to loss ofseal/recording from other micro-pipettes that are connected to cells. INother circumstances, probes can already be on/close to the test target,and vibrating and shaking the tip can damage the test target. This, ofcourse, can also be important to scalability and overall throughput.Automating the micro-pipette/probe exchange is technically difficult andexpensive, which makes the present cleaning step useful in practice andgate-keeper for automations.

The preparation of the micro-pipette/-electrode/-probe automatically issubstantially faster than micro-pipette/-electrode/-probe exchange, andthus increases throughput.

The present invention's speed/increased efficiency versus theconventional way of changing the probe is another benefit of the presentinvention.

In another exemplary embodiment of the present invention, a system forstimulating and/or measuring one or more physical characteristics of atest target located in a test area comprises detecting one or morecharacteristics of test targets comprising an automatedmicro-/nano-positioner such as a micromanipulator capable of moving theapertured micro-pipette/-electrode/-probe tip to the surface of testtargets located in the test area and to a separate cleaning area by theagency of an automated process, the micro-pipette/-electrode/-probe hasan apertured surface suitable for forming quality contact and interfacewith the test target as characterized by electrical, mechanical,chemical or other physical variables, a re-use assembly comprising asample holder with a test area for test targets and a separate cleaningarea configured to contain cleaning solution, an automated pressureassembly in communication with the micro-pipette/-electrode/-probe thatcan, via the aperture, control the pressure applied to the test targetfrom negative to positive pressures relative to the atmosphere to drawtoward and expel from the apertured surface and substantially immediateinner surface(s) of the tip behind the aperture at least a portion ofcleaning solution, and wherein preparing the apertured surface andsubstantially immediate inner surface of the tip behind the aperturedsurface enables re-use of the same micro-pipette/-electrode/-probe withat least two consecutive and different test targets.

Preparing the apertured surface and substantially immediate innersurface(s) of the tip behind the apertured surface can further compriserinsing by moving the tip after cleaning to a test area location awayfrom next test target or to a separate rinse area with rinse solutionthat is not harmful to the test target, and applying at least onepositive or negative pressure pulse.

The system can include processors/memory/software (via a separatecontrol apparatus, PC or control that is integrated to some of otherapparatus' controllers included in the system like a micromanipulator orpressure controller).

The cleaning solution can be a detergent suitable to remove thecontaminants and debris relevant to a specific test, which may includeorganic and inorganic substances, enzymes or other substances that aregenerally used for removing such contaminant and debris.

The micro-pipette/-electrode/-probe has an aperture/opening in the endand a channel or equivalent structure through it to enableconveying/mediating pressure via the aperture to the test target, aswell as, via the same aperture, stimulate and/or measure electrical,mechanical, chemical or other types of physical quantities from the testtarget.

Patch clamp recording is but one example where a hollow glassmicro-pipette is used to stimulate and measure electrically from a cell.The micro-pipettes usually are made from glass with a tip that is fromsub-micrometer to tens of micrometers in size, and has a smooth surface.When the tip is clean, it seals tightly with the cell surface. Qualityof the contact and interface (“seal”) can be measured electrically, anda good quality can range from tens to hundreds of megaohms to more thana gigaohm can be had depending on the specific type of test mode.

A goal of the invention is to enable re-use of the same aperturedsurface on two or more test targets. To do this, the apertured surfacemust of course be clean enough to form high-quality contact andinterface with the test target to enable the testing of electrical,mechanical, chemical or other types of physical variables relevant to aplanned test.

The size and shape of the aperture, as well as its surface properties,are suitable to form such high-quality contact and interface, and theaperture must be sufficiently clean from contaminants and debris inorder to be re-used.

The route through the micro-pipette/-electrode/-probe via aperture tothe test target should be substantially free of occlusions to allow forreliable and high-quality interfaces to the test target for pressurecontrol and for stimulation and/or measurement of the physical quantity.Cleaning does not only clean the apertured surface, but also the tiparea behind it to clean the tip inner wall surface.

The present invention presents systems and in-situ methods foreffectively removing biological debris from patch-clamp electrodes,including conventional glass pipettes and planar patch chips, to enablethem to repeatedly re-used.

In exemplary embodiments, effectively removing biological debris isaccomplished via cleaning the area where the biological debris islocated. This can be done with a cleaning step, a cleaning step and arinsing step, and with other methods of effectively removing biologicaldebris from proximity of the aperture that forms the seal so the samesealing assembly can be re-used to make another seal with a differentcell.

While the present invention refers to systems and methods enablingpatch-clamp re-use, and particularly pipettes and planar patch chips, itwill be understood by those of skill in the art that the presentinvention can apply to other environments where, for example, aperturedsurfaces that are in proximity to or in contact with biologicalmaterial, like cell membranes, can benefit from an inventive way ofeffectively removing biological debris so the same apertured surface canbe used once again.

Thus, in some exemplary embodiment, the present invention refers tosystems and methods enabling re-use of an apertured surface that isexposed to biological material(s). In a particular embodiment, theapertured surface is the aperture of a pipette used in patch-clampsystems and methods. The problem of continuingly exchanging apatch-clamp electrode for a fresh one can be circumvented by removingcellular debris from a previously used electrode aperture, thus enablingthe same electrode to be used again. The present invention can utilizean electrode cleaning step that enables the re-use of a single electrodein multiple sequential patch-clamp trials, and commoditizes the use ofcustom-geometry electrodes. The present invention thus reduces a user'sexpenses associated with electrodes and intra-electrode electrolyticsolution.

In another particular embodiment, the apertured surface is one or moreof the many apertures of planar patch chips.

In an exemplary embodiment, the present invention effectively removesbiological debris from the apertured surface so it can be re-used toachieve gigaseals. In another exemplary embodiment, it is understoodthat not every surface needs such extreme cleaning. For example, in someconventional planar patch-clamp systems, to be effective, the seal ofresistance can be ≤1 GΩ, for example 20-250 MΩ. The present invention iseffective in such systems to enable re-use of the apertured surfaces ofthis kind of planar patch-clamp systems.

The present invention provides a technique for re-using planarpatch-clamp chips where the cleaning step does not require the planarchip to be manually extracted from the patch-clamp device to bechemically treated and dried.

The present invention provides a re-use technique applicable to thedelicate electrode tip of a patch-clamp pipette. The aperture ofpatch-clamp electrodes are generally from 0.5-10 μm, much smaller thanthat of capillaries used for electrophoresis (20-200 μm), making thepresently inventive methods distinct from the art of cleaningcapillaries used for electrophoresis.

In an exemplary embodiment, the present invention comprises a method ofdetecting one or more characteristics of cells comprising forming aresistance seal with a membrane of a cell with an apertured surface,forming a resistance seal with a membrane of a different cell with theapertured surface, and preparing the apertured surface, whereinpreparing the apertured surface enables re-use of at least portions ofthe same apertured surface with the different cells.

In an exemplary embodiment, pipettes and planar patch chips are re-used(apertured surfaces are re-used) via cleaning. Other systems and methodsof clearing biological debris from the proximity of the aperture toenable re-use are contemplated. For example, apertured surfaces can betreated with a gas, heated, etched with an ion beam, melted andreformed, sputter coated, or otherwise treated to enable re-use of thesame apertured surface with a number of different cells.

Some conventional planar patch-clamp systems only achieve a loose-sealrecording that amounts to a seal of resistance≤1 GΩ, for example 20-250MΩ. While this does not enable high-resolution whole-cell recording, isstill useful for studying ion channels. The present invention remainsuseful in such planar patch-clamp embodiments to enable re-use of one ormore of the apertured surface(s) with different cells, even if the sealof resistance≤1 GΩ.

In other exemplary embodiments, the resistance of each resistance sealcan be ≥1 GΩ.

At least portions of the same apertured surface can form a resistanceseal with a membrane of at least five different cells, and wherein theresistance of each resistance seal is ≥1 GΩ.

At least portions of the same apertured surface can form a resistanceseal with a membrane of at least seven different cells, and wherein theresistance of each resistance seal is ≥1 GΩ.

Preparing the apertured surface can occur in-between each step offorming a resistance seal with a membrane of a cell.

Preparing the apertured surface can comprise subjecting the aperturedsurface to a cleaning solution.

Preparing the apertured surface can comprise subjecting the aperturedsurface to a cleaning solution and a rinse solution.

The apertured surface can comprise the aperture of a pipette.

The apertured surface can comprise the aperture in a planar electrode.

The aperture of the pipette or planar electrode can have a diameterbetween 0.5-10 μm.

In another exemplary embodiment, the present invention comprises amethod of repeatedly electronically isolating currents measured across amembrane of a cell comprising forming a resistance seal with a membraneof a cell with an apertured surface, repeating the step of forming aresistance seal with a membrane of a cell with at least seven differentcells with the same apertured surface, and preparing the aperturedsurface at least once in-between two consecutive steps of forming aresistance seal with a membrane of a cell, wherein preparing theapertured surface enables re-use of the same apertured surface with thedifferent cells, and wherein each resistance seal is ≥1 GΩ.

In another exemplary embodiment, the present invention comprises systemfor detecting one or more characteristics of cells comprising anapertured surface used for forming a resistance seal with a membrane ofa cell, and a re-use assembly, wherein the re-use assembly prepares atleast portions of the apertured surface so it can be re-used to form aresistance seal with a membrane of at least two different cells.

The system can further comprise a suction assembly in communication withthe apertured surface and providing a resistance for each resistanceseal of ≥1 GΩ.

The re-use assembly can comprise a cleaning solution.

The re-use assembly can comprise a rinse solution.

In another exemplary embodiment, the present invention is a method ofremoving biological debris from a patch-clamp electrode comprising oneor more chemical treatments applied sequentially to wash the inner andouter surfaces of at least a portion of the electrode by the agency ofpneumatic pressures applied to the electrode lumen.

The patch-clamp electrode can be a micro-pipette formed of a hollowglass capillary pulled to a fine tip with an aperture diameter between0.5-10 μm at its distal end.

The patch-clamp electrode can be a planar patch chip.

One of the chemical treatments can comprise a composition comprisingsodium bicarbonate, sodium (C10-C16) linear alkylbenzene sulfonate(LAS), sodium tripolyphosphate, tetrasodium pyrophosphate, sodiumcarbonate, and sodium alcohol sulfate dissolved in deionized water.

A final chemical treatment can be non-cytotoxic. The final chemicaltreatment can be buffered solution where the pH is within the range 7.2to 7.5 and the osmolarity is within the range 290-320.

The agency of pneumatic pressures can comprise alternating positive andnegative pressures (up to ±1,500 mbar) relative to atmosphere to bringthe cleaning solution in contact with the electrode interior surface fora specified time interval.

The application of pneumatic pressures can be controlled by a computeror microcontroller.

The electrode can be moved between chambers containing chemicaltreatments using a manipulator, for example, a three-axismicromanipulator.

In an exemplary embodiment, a first aCSF (artificial cerebrospinalfluid) wash is performed (−200 mbar-10 seconds, 200 mbar-30 seconds), acleaning agent is cycled preferably six times (−200 mbar-10 seconds, 200mbar-30 seconds), and a second aCSF wash is performed (−200 mbar-10seconds, 200 mbar-30 seconds).

In another exemplary embodiment, the present invention comprises systemsand methods for cleaning patch-clamp glass pipette electrodes thatenable their re-use. By immersing pipette tips into a detergent,followed by rinsing, pipettes were re-usable at least ten times withlittle to no degradation in signal fidelity, in experimentalpreparations ranging from human embryonic kidney cells to neurons inculture, slices, and in vivo.

In another exemplary embodiment, pipette tips are immersed into aconcentrated, anionic detergent for manual and ultrasonic cleaning, forexample, Alconox® (Alconox Inc) brand detergent, at 2% w/v (20 mg/ml),for 60 seconds, which permits multiple re-uses in cell cultures, tissueslices, and in vivo. There was no detectable Alconox® in the pipetteafter cleaning, and ion channel pharmacology results wereindistinguishable between fresh and cleaned pipettes. The presentpipette cleaning method for the first time enables dramatically improvedautomation of patch-clamp, as demonstrate with unattended, sequentialpatch-clamp recordings in cell culture and in vivo.

These and other objects, features and advantages of the presentinvention will become more apparent upon reading the followingspecification in conjunction with the accompanying drawing figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying Figures, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1A is a schematic of a conventional patch-clamp, modified to enablethe present process according to an exemplary embodiment. FIG. 1B is aschematic of a planar patch-clamp embodiment, modified to enable thepresent process according to an exemplary embodiment.

FIG. 2A shows a Scanning Electron Microscopy (SEM) image of a freshpipette. FIG. 2B shows an SEM image of a pipette following a traditionalpatch-clamp trial. FIG. 2C shows an SEM image of a pipette following atraditional patch-clamp trial and a subsequent application of thecleaning method according to an exemplary embodiment of the presentinvention.

FIG. 3 is a graph of the gigaseal resistances of ten sequentialpatch-clamp trials using one pipette that was cleaned according to anexemplary embodiment of the present invention.

FIGS. 4A-D illustrate that during a whole-cell patch-clamp recording,cell membrane bonds to the inner walls of the pipette (FIG. 4A). Aschematic of the present process according to an exemplary embodiment(FIG. 4B). Representative gigaseal formation traces (FIG. 4C).Representative resistances (FIG. 4D).

FIG. 5 presents three representative gigaseals obtained with freshpipettes that were pre-cleaned in Alconox®. Pre-cleaning did not hinderthe formation of stable gigaseals.

FIGS. 6A-D illustrate recording quality parameters R_(GS), T_(GS) andR_(a) (whole-cell access resistance) (FIG. 6A). Pipette resistancebefore and after cleaning with Alconox® (FIG. 6B). Change in pipetteresistance from first to tenth re-use (FIG. 6C). Representativewhole-cell responses to step current injections in differentexperimental preparations (FIG. 6D).

FIG. 7 is a graph of gigaseal resistances of thirty re-use attempts witha single pipette over time.

FIG. 8 illustrates graphs for five pipettes wherein successful attemptsresulting in whole-cell recordings are shown.

FIGS. 9A-C illustrate that LAS is the prevalent cytotoxic ingredient inAlconox®. FIG. 9A illustrates the chemical formula for sodium (C10-C16)linear alkylbenzene sulfonate (LAS). How the contents of cleanedpipettes were collected in vials (FIG. 9B). ESI-MS spectrum (FIG. 9C).

FIG. 10 is a schematic ESI-MS spectrum characterization of pipettescontaining 67 μg/ml Alconox® dissolved in DI water. The main cytotoxiccomponents of Alconox®, C10-C12 LAS compounds were identified in thesolution (C10: expected m/z: 297.1, found: 297.1; C11: expected m/z:311.2, found: 311.1; C12: expected m/z: 325.2, found: 325.2).

FIGS. 11A-D illustrate pipette cleaning in conjunction with GABA_(A)Rpharmacology. Representative current traces recorded by whole-cellpatch-clamp recording of HEK293T cells transfected with α1β2γ2sGABA_(A)Rs (FIG. 11A). Representative normalized peak current responsesto different GABA concentrations (FIG. 11B). Dose responsecharacteristics of cells patched with fresh and re-used pipettes (FIG.11C). That a low dose of Alconox® (67 μg/mL, or equivalently, 385×thedetection limit of remaining Alconox® in the pipette after cleaning) inthe internal solution does not affect dose response characteristics(FIG. 11D).

FIGS. 12A-B show a pipette containing a high dose (10 mg/mL) of Alconox®damages the target cell during a patch attempt. FIG. 12A: A gigasealfails to form after >3 min, suggesting that Alconox® is destroying cellmembrane. FIG. 12B: Cell apoptosis during the gigaseal process isevident. Arrow indicates blebs forming on the cell. Scale bar: 10 μm.

DETAIL DESCRIPTION OF THE INVENTION

To facilitate an understanding of the principles and features of thevarious embodiments of the invention, various illustrative embodimentsare explained below. Although exemplary embodiments of the invention areexplained in detail, it is to be understood that other embodiments arecontemplated. Accordingly, it is not intended that the invention islimited in its scope to the details of construction and arrangement ofcomponents set forth in the following description or illustrated in thedrawings. The invention is capable of other embodiments and of beingpracticed or carried out in various ways. Also, in describing theexemplary embodiments, specific terminology will be resorted to for thesake of clarity.

It must also be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,reference to a component is intended also to include composition of aplurality of components. References to a composition containing “a”constituent is intended to include other constituents in addition to theone named.

Also, in describing the exemplary embodiments, terminology will beresorted to for the sake of clarity. It is intended that each termcontemplates its broadest meaning as understood by those skilled in theart and includes all technical equivalents which operate in a similarmanner to accomplish a similar purpose.

Ranges may be expressed herein as from “about” or “approximately” or“substantially” one particular value and/or to “about” or“approximately” or “substantially” another particular value. When such arange is expressed, other exemplary embodiments include from the oneparticular value and/or to the other particular value.

Similarly, as used herein, “substantially free” of something, or“substantially pure”, and like characterizations, can include both being“at least substantially free” of something, or “at least substantiallypure”, and being “completely free” of something, or “completely pure”.

By “comprising” or “containing” or “including” is meant that at leastthe named compound, element, particle, or method step is present in thecomposition or article or method, but does not exclude the presence ofother compounds, materials, particles, method steps, even if the othersuch compounds, material, particles, method steps have the same functionas what is named.

It is also to be understood that the mention of one or more method stepsdoes not preclude the presence of additional method steps or interveningmethod steps between those steps expressly identified. Similarly, it isalso to be understood that the mention of one or more components in acomposition does not preclude the presence of additional components thanthose expressly identified.

The materials described as making up the various elements of theinvention are intended to be illustrative and not restrictive. Manysuitable materials that would perform the same or a similar function asthe materials described herein are intended to be embraced within thescope of the invention. Such other materials not described herein caninclude, but are not limited to, for example, materials that aredeveloped after the time of the development of the invention.

As shown in FIG. 1A, in an exemplary patch-clamp embodiment of thepresent invention, the distal end (an apertured surface) of a glasspipette 1 is attached to a single cell 2 in a preparation area 3. Thepipette 1 is attached at the proximal end to a pipette holder 4. Acleaning solution chamber 7 containing a cleaning solution and a rinsesolution chamber 8 containing rinse solution are situated near thebiological preparation area 3. Pressure is applied via a pressurecontroller 5 to the pipette 1 through the pipette holder 4. A three-axismicromanipulator 6 moves the pipette 1 and the pipette holder 4 in threedimensions.

As shown in FIG. 1B, in a planar patch-clamp embodiment of the presentinvention, a cell 9 in an extracellular fluid medium 14 is sealed to (atan apertured surface of) a planar glass electrode 10 at the opening tothe intracellular fluid space 11. Pressure is applied via one channel ona pressure controller 12. Cleaning fluid from cleaning fluid chamber 13is dispensed into the intracellular fluid space 11 via another channelon the pressure controller 12.

In operation of the system of FIG. 1A, the micromanipulator 6 is used tobring the glass pipette 1 to the preparation area 3 to approach thesingle cell 2. Negative pressure is applied via the pressure controller5 and the membrane of the cell 2 adheres to the distal end of thepipette 1.

Following an electrophysiological experiment, the micromanipulator 6withdraws the pipette 1 from the cell 2 and expels large, adherentcellular debris from the pipette 1 with high positive pressure from thepressure controller 5. The micromanipulator 6 then moves the pipette 1,under high positive pressure via the pressure controller 5, from thepreparation area 3 and into the chamber 7 containing cleaning solution.Pressure is then cycled a number of times between positive pressure andnegative pressure with the pressure controller 5 to bring the cleaningsolution within the chamber 7 into contact with the interior andexterior surfaces of the pipette 1 and to cyclically apply shear forcesto the interior surface of the pipette 1. The micromanipulator 6 thenmoves the pipette 1, under high positive pressure from the pressurecontroller 5, from the chamber 7 containing cleaning solution into thechamber 8 containing rinse solution. Pressure is then cycled a number oftimes between positive pressure and negative pressure with the pressurecontroller 5 to bring the rinse solution within the chamber 8 intocontact with the interior and exterior surfaces of the pipette 1 and toremove any residual cleaning solution from the interior and exteriorsurfaces of the pipette 1. The micromanipulator 6 then moves the pipette1, under high positive pressure from the pressure controller 5, from thechamber containing rinse solution 8 and back to the preparation area 3.Subsequent electrophysiological trials can be performed by repeating thesteps described above.

In operation of the system of FIG. 1B, the suspension containing one ormore cells 9 in the extracellular fluid medium 14 is passed over anaperture in the planar glass electrode 10 leading to the channel 11containing intracellular fluid. Suction is applied to the intracellularfluid space 11 via pressure controller 12 to draw the suspended cell 9to the aperture and form a gigaseal.

Following the electrophysiological experiment, high pressure is appliedto the intracellular space 11 via pressure controller 12 to eject thecell 9 and bulk cellular debris from the aperture. Pressure is appliedto the cleaning solution channel 13 via pressure controller 12 to ejectcleaning fluid into the intracellular space 11 and bring cleaning fluidinto contact with the interior wall of the intracellular channel 11.Simultaneously, pressure is cycled between positive and negativepressure (suction) on the intracellular channel 11 a number of times tocyclically apply shear forces to the surface of intracellular channel11. The cleaning solution channel 13 is then returned to atmosphericpressure or slight negative pressure via the pressure controller 12 toprevent excess cleaning solution from entering the intracellular space11. Positive pressure is applied to the intracellular space 11 viapressure controller 12 to eject any remaining debris and residualcleaning chemical. Subsequent electrophysiological trials can beperformed by repeating the steps described above.

FIG. 2 are SEM images of pipette tips. FIG. 2A is an SEM image of afresh pipette. FIG. 2B shows an SEM image of a pipette tip cleaned withaCSF and being visibly contaminated with cell membrane residue,illustrating the necessity of throwing out the conventional pipetteafter each use (it cannot be re-used without a viable cleaning andrinsing regime heretofore unknown).

FIG. 2C is an SEM image of a pipette following the present inventivecleaning and rinsing regime (namely using Alconox®), illustrating thecapability of pipette re-use. The Alconox®-cleaned pipette tip resemblesthat of the fresh pipette. Pipette filament denoted with *. Scale bar: 1μm.

FIG. 3 illustrates the resulting gigaseal resistances of ten sequentialpatch-clamp trials using one pipette that was cleaned with the presentmethod. In 8/10 trials, a gigaseal was successfully established.

In exemplary embodiments of the present invention shown in FIGS. 4-12,FIGS. 4A-D illustrate cleaning patch-clamp pipettes. During a whole-cellpatch-clamp recording, cell membrane bonds to the inner walls of thepipette (FIG. 4A). After the recording is terminated, membrane residueremains, preventing the pipette from being used for subsequentrecordings. (FIG. 4B) to clean, (i) the pipette is moved from theexperimental preparation to a wash bath where a cleaning agent is cycledwithin the tip, (ii) then to a rinse bath where the remaining cleaningagent is expelled into aCSF, (iii) and returned back to the experimentalpreparation. (FIG. 4C) Representative gigaseal formation traces. Whenusing contaminated pipettes cleaned with Alconox®, a multi-GΩ seal formsreliably, as would be expected when using a fresh pipette. On the otherhand, cleaning with aCSF and bleach does not result in gigasealformation. R_(GS): maximum gigaseal resistance, T_(GS): time (s) toreach 1 GΩ (horizontal dashed line). (FIG. 4D). Of the six testeddetergents and aCSF, only Alconox® reliably achieved gigasealresistances comparable to those of fresh pipettes (p<0.001; one-wayANOVA with Dunnett's post-hoc test. *: p<0.001; n.s.: not significant,p>0.9). Data shown as mean±s.d.; n for each cleaning agent is shown inparentheses. PLA2: Phospholipase A2; SDS: Sodium Dodecyl Sulfate.

As discussed above, during a whole-cell patch-clamp recording (FIG. 4A),cell membrane bonds to the inner walls of the pipette. After therecording is terminated, membrane residue remains, preventing thepipette from being used for subsequent recordings. After a recordingattempt in an experimental preparation, pipette cleaning is accomplishedin three steps (FIG. 4B). First, the pipette is moved to a bathcontaining a cleaning agent. Using an electronic pressure regulator, aseries of alternating negative and positive pressures is applied to thepipette to cycle the cleaning agent within the tip.

Second, the pipette is moved to a bath containing aCSF. Using anotherseries of pressure pulses, any remaining cleaning agent in the pipetteis expelled. Based on the calculated fluid flow rates from pipettes (˜3nL/s, based on pipette inner diameter of 1 μm), this pressure series issufficient to intake the cleaning agent at least 200 μm into the lumenof the pipette. This exceeds the height of a typical lipid ‘bleb’ thatforms in the pipette tip during a gigaseal (30-60 μm).

Lastly, the pipette is moved back to the experimental preparation.Pipettes can then be immediately re-used for a subsequent patch-clampattempt. Refilling a pipette with internal solution is unnecessarybecause pipettes are commonly filled with enough solution for hundredsof attempts. All moving and cleaning steps together require 60 seconds.

TABLE 1 Cleaning Solution Source Bleach (8.25% Sodium The Clorox Companyhypochlorite) Triton X-100 X100-5 mL (Sigma-Aldrich) Acetone BDH1101(VWR) Phosopolipase A₂ P7778 (Sigma-Aldrich) (PLA, 0.0001% w/v in 10 mMHEPES buffer) Sodium TCD0990 (VWR) Dodecylbenzenesulfonate (SDS, 1% w/v)Alconox ® (2% w/v) Alconox Inc.

Alconox® was one cleaning agent that enabled pipettes to be re-used inquantities sought. Whole-cell patch-clamp recordings on human embryonickidney (HEK293T) cells were performed using fresh (previously unused)pipettes, cleaned them with one of six different commonly availableglassware cleaning agents as shown in TABLE 1, as well as a controlsolution (aCSF), and attempted to patch another cell with the samepipette. On the second attempt, pipettes cleaned with Alconox®, but notwith any other cleaning agent, produced gigaseal resistances (R_(G)s)that were not significantly different from those produced by freshpipettes (FIG. 4CD). Successful whole-cell recording rates were notdifferent between fresh and Alconox®-cleaned pipettes (fresh=92.6%,Alconox®-cleaned=88.9%, p=0.87, Fisher's exact test; other detergentsand aCSF=0%).

Gigaseals were held for 1-2 minutes to ensure stability. No gigasealswere spontaneously lost during this time in either fresh orAlconox®-cleaned pipettes. When using Alconox®-cleaned pipettes, anoutside-out patch reliably formed after withdrawing the pipette from thecell, as would be expected if using fresh pipettes. Further,pre-cleaning fresh pipettes in Alconox® had no effect on gigasealformation. FIG. 5 presents three representative gigaseals obtained withfresh pipettes that were pre-cleaned in Alconox®. Pre-cleaning did nothinder the formation of stable gigaseals.

Using SEM, the tip of the pipettes was confirmed as being cleaned usingAlconox® but not aCSF (FIGS. 2A-C). Unless stated otherwise, allsubsequent experiments used Alconox®.

FIGS. 6A-D illustrate that pipettes can be successfully re-used tentimes. (FIG. 6A) Recording quality parameters R_(GS), T_(GS) (defined inFIG. 4C) and R_(a) (whole-cell access resistance) do not decrease overten re-uses of the same pipette (n=8 pipettes). Dashed line: 1 GΩ(threshold for gigaseal). Gray circles: individual trials; blackcircles: representative experiment with a single pipette. Data shown asmean±s.d. (FIG. 6B) Pipette resistance before and after cleaning withAlconox® (n=88 pairs, 8 pipettes). (FIG. 6C) Change in pipetteresistance from first to tenth re-use. After ten re-uses, resistancechanged (ΔR) by median: 0.175 MΩ, max: 0.61 MΩ, min: −0.44 MΩ from freshpipette. (FIG. 6D) Representative whole-cell responses to step currentinjections in different experimental preparations. Recordings fromHEK293T cells were obtained from the representative experiment (blackcircles) in (FIG. 6A). In each set, a single pipette is used for allthree whole-cell recordings. In all preparations pipettes were re-usedup to ten times.

As discussed above, pipettes could be re-used ten times consecutivelywith little to no degradation in recording quality if they were cleanedbetween each patch-clamp attempt (FIG. 6A). 84 HEK293T cells weresuccessfully patched (of n=88 attempts including one fresh and 10 ten;success rate=95%) using eight pipettes, and found no effect of thenumber of re-uses on gigaseal resistance (R_(G)s), the time to reach 1GΩ (T_(GS)), and the access resistance of the patched cell (R_(a)) foreach trial (one-way repeated-measures ANOVA with number of re-uses(1-10) as predictor; R_(GS): F_(10,40)=0.99, p=0.46, n=8; T_(GS):F_(10,40)=0.36, p=0.96, n=5, R_(a): F_(10,40)=0.72, p=0.70, n=5).

Notably, even when pipettes failed to reach a gigaseal (e.g., R_(GS)<1GΩ on third, fourth re-use in FIG. 6A), they were successfully cleanedand re-used again, suggesting that gigaseal failures did notirreversibly contaminate the pipette tip. Beyond ten re-uses were nottested to perform a systematic study of pipette longevity; however, apilot experiment confirmed the suspicion that pipettes could not bere-used indefinitely, when three consecutive failed patch-clamp attemptsoccurred after 26 re-uses.

FIG. 7 illustrates the gigaseal resistances of thirty re-use attemptswith a single pipette. The pipette was successfully re-used twenty-sixtimes (green circles: R>1 GΩ). The pipette did not reach a gigaseal onthe fifth re-use, and consecutively on twenty-seventh through thirtiethre-use (circles: R<1 GΩ). Gigaseal attempts are coded from light to darkaccording to the number of times the pipette was used. Afterapproximately fourteen re-uses, the time to form a gigaseal increases.

The patency of the pipette tip is commonly assessed during patch-clamprecording by measuring its electrical resistance. To assess whethercleaning reduced the amount of contamination on the pipette tip, theresistance was measured twice: once immediately after completing awhole-cell recording (before cleaning) and once more after cleaning. Asexpected, pipette resistance before cleaning was higher than after (FIG.6B; before: median=5.93 MΩ, range: 4.49 to 15.0 MΩ; after: median=5.77MΩ, range=4.46 to 6.28 MΩ; p<0.05, Wilcoxon signed-rank test). Over 10re-uses, the resistance of the pipette did not change significantly(FIG. 6C); p>0.19 for first-tenth re-use, n=8, Wilcoxon signed-ranktest). Together, these results suggest that cleaning eliminates residueobstructions on the pipette tip over ten re-uses.

Repeated patch-clamping in neuron cultures, acute brain slices, and invivo were done using a single pipette for each experimental preparation(FIG. 6D). Cleaning did not visibly alter action potential generation inthese experiments. In brain slices, successful gigaseals and successfulwhole-cell recordings were attained at similar rates between fresh andcleaned pipettes (gigaseals: fresh=78%, cleaned=82%, p=0.73; whole-cellrecordings: fresh=39%, cleaned=48%, p=0.58, Fisher's exact test).

As shown in FIG. 8, in slices, neurons were recorded and found to havethe access resistance stable across that time, indicating thatwhole-cell recordings obtained using cleaned pipettes are stable overthe duration of a typical experiment. For each plot, a single pipettewas used. Only successful attempts (resulting in whole-cell recordings)are shown. Cells were held for ˜20 minutes. Access resistance did notincrease for fresh pipettes as well as for pipettes re-used 1-10 times,indicating stable whole-cell recordings. In vivo, the Autopatcher wasused to patch-clamp repeatedly with the same pipette in mouse barrelcortex (depths: 400-600 um).

Verifying Detergent Removal after Pipette Cleaning

A major concern of using any detergent to clean pipettes is thatresidual surfactants in the pipette could damage the cell or affect itsnormal biophysical activity during a cell-attached or whole-cellpatch-clamp recording. For example, Alconox® comprises 33-43% sodiumbicarbonate, 10-20% sodium (C10-C16) LAS, 5-15% sodium tripolyphosphate,5-15% tetrasodium pyrophosphate, 1-10% sodium carbonate, and 1-5% sodiumalcohol sulfate. LAS was chosen, a commonly-used surfactant, as a proxyfor measuring Alconox® concentration remaining after rinsing.

FIGS. 9A-C illustrate detection of LAS in cleaned pipettes usingelectrospray ionization mass spectrometry (ESI-MS). (FIG. 9A) LAS is theprevalent cytotoxic ingredient in Alconox®. Alconox® is composed of LASwith carbon chains lengths 10-12 (C10-C12). (FIG. 9B) The contents ofcleaned pipettes were collected in vials. Known amounts of Alconox® wereanalyzed to find the detection limit. (FIG. 9C) ESI-MS spectrum. C10-C12LAS is not detectable in cleaned pipettes (top) but is detected in adefined Alconox® solution (174 ng/mL Alconox® in DI water, bottom),indicating that less than 174 ng/mL of Alconox® remains in the pipettesafter cleaning. C10: expected m/z: 297.1, found: 297.3; C11: expectedm/z: 311.2, found: 311.2; C12: expected m/z: 325.2, found: 325.1.

As discussed above, the amount of Alconox® remaining after cleaning wasmeasured using electrospray ionization mass spectrometry (ESI-MS). Asshown in FIG. 10, only C10-C12 LAS was found in significant quantitiesin the Alconox® solution, so the analysis was focused on this family ofcompounds (FIG. 9A). No traces of LAS was found in cleaned pipettes(FIG. 9B, C (top)), while the instrumentation detection limit was foundto be 174 ng/mL (FIG. 9C (bottom)). Thus, less than 174 ng/mL ofAlconox® remained in the pipettes after the cleaning procedure. As acontrol, as shown in FIG. 10, LAS was ensured it could still be detectedusing ESI-MS when purposefully introduced in small concentrations intothe pipette.

Any effect of trace amounts of Alconox® in cleaned pipettes on cellreceptor pharmacology would be highly undesirable. Residual LAS coulddisrupt gigaseal formation, thus decreasing signal quality or, moresubtly, interact with amphipathic allosteric modulatory pockets onreceptors, thus covertly compromising pharmacological experiments. Theγ-aminobutyric acid type A Receptor (GABA_(A)R) is highly sensitive toextracellular application of surfactants, including LAS; itsintracellular effects have not been thoroughly studied. Nevertheless, itis reasoned that GABAAR could serve as an indicator of adverse effectsof Alconox®.

FIGS. 11A-D illustrate pipette cleaning in conjunction with GABAARpharmacology. (FIG. 11A) Representative current traces recorded bywhole-cell patch-clamp recording of HEK293T cells transfected withα1β2γ2s GABA_(A)Rs. Black bar denotes GABA application (FIG. 11B)Representative normalized peak current responses to different GABAconcentrations. The response captured with a fresh pipette is similar tothat captured with a used pipette (fourth re-use). (FIG. 11C) Doseresponse characteristics of cells patched with fresh and re-usedpipettes. i_(pk): peak evoked current; h: Hill coefficient; EC₅₀:half-maximal response concentration. No change in the threecharacteristics is observed over four re-uses. (FIG. 11D) A low dose ofAlconox® (67 μg/mL, or equivalently, 385×the detection limit ofremaining Alconox® in the pipette after cleaning) in the internalsolution does not affect dose response characteristics.

As discussed above, HEK293T cells expressing GABAAR were used as a modelsystem to verify that cleaned pipettes are pharmacologically inert.Patch-clamping using fresh and cleaned pipettes was performed, and fitthe measured whole-cell current responses to increasing concentrationsof GABA (FIG. 11A) to the Hill equation. Neither the peak evoked current(I_(pk)), the Hill's coefficient (h), nor the half maximal effectiveconcentration (EC₅₀) metrics changed as a function of re-uses (linearregression model, H₀: slope≠0; I_(pk): p=0.998, 95% CI: −318 to 317 pA;h: p=0.719, 95% CI: −0.065 to 0.093; EC₅₀: p=0.751, 95% CI: −6.75 to4.90).

This finding demonstrates that GABA concentration-response curves of thepatch-clamped cells do not change on a population level as a function ofthe number of times a pipette has been re-used (FIGS. 11B, C). Overall,pharmacology results obtained using fresh pipettes wereindistinguishable from those obtained using cleaned pipettes, suggestingno effect of trace amounts of Alconox® on normal receptor function ofGABA_(A)Rs.

It was also found that even if small amounts of Alconox® remain in thepipette after cleaning, they do not impact gigaseal formation orGABA_(A)R pharmacology. When 67 μg/mL of Alconox® (385× the ESI-MSdetection limit) was intentionally dissolved in the pipette internalsolution, gigaseals were still reliably achieved in all tested pipettes(n=9). No pharmacological difference between these pipettes and freshones was detected (FIG. 11D; I_(pk): p=0.37; h: p=0.99; EC₅₀: p=0.19).On the other hand, when a much larger dose, 10 mg/mL Alconox® (0.5×theconcentration in the wash bath) was added to the pipette, gigaseals didnot form and the targeted cell exhibited clear apoptotic blebbing. See,FIGS. 12A-B.

Unattended, Sequential Patch-Clamp Recording

Previous efforts have partially or fully automated the process ofobtaining a single whole-cell recording in vitro as well as in vivo;however, in all of these studies, a trained user was needed to exchangepipettes to start another attempt. Removing this manual step wouldenable unattended patch-clamp experiments and improve scalability andthroughput. To explore this, the pipette cleaning algorithm wasintegrated into the previously developed Autopatcher software. Theresulting robot was tested, which was called ‘patcherBot’ by firstperforming patch-clamp recordings in HEK293T cells.

After the user selected candidate cells for patch-clamp recording, thepatcherBot obtained successful gigaseals in 9/10 attempts, andsuccessful whole-cell recordings in 6/10 attempts over the span of 33minutes of unattended operation using a single pipette. The patcherBotwas also deployed to perform blind, in vivo patch-clamp recordings inthe mouse barrel cortex. Using four pipettes, the robot obtainedsuccessful gigaseals in 13/34 attempts, and successful whole-cellrecordings in 10/34 attempts over a total span of 171 minutes in vivo(FIG. 6D).

Discussion

Discovering that pipettes could be reliably cleaned and re-used multipletimes was surprising given the dogmatic, decades-long practice ofreplacing pipettes after each patch-clamp attempt. The present simple,fast, and automated procedure comprises dipping pipettes into acommercially-available detergent, Alconox®, followed by rinsing in aCSF.It is herein demonstrated the effectiveness of this cleaning method byre-using pipettes ten times, with no decrease in whole-cell recordingquality in cultured cells, acute brain slices, and in vivo. Aftercleaning, the residual Alconox® mass concentration in the pipettes wasquantified to be less than 174 ng/mL, and shown to have nopharmacological effect on GABA_(A)Rs. Since pipette cleaning isautomatic, it was integrated into the Autopatcher to perform unattended,sequential patch-clamp recordings on HEK293T cells in vitro and neuronsin vivo.

Alconox® is composed of a surfactant, emulsifier and water softener, andin solution, it interacts with cell membranes bound to the glass pipettetip. These three ingredients solubilize adhered lipids, stabilize thelipid micelles in solution, and enhance surfactant effectiveness,respectively. After testing various cleaning agents, it was not evidentwhy only Alconox® sufficiently cleaned pipettes to enable their re-use.Interestingly, prior studies used bleach to successfully clean planarborosilicate patch-clamp chips up to five times for whole-cellrecording; yet in the present experiments using conventional pipettes,bleach did not work, suggesting that the geometry of the experimentalpreparation may influence cleaning effectiveness.

Overall, the precise mechanism of how cell membranes bond with glassduring a gigaseal is still not understood despite notable efforts,making it difficult to devise a cleaning strategy from basic principles.A detailed biochemical study of gigaseal formation could thereforegreatly inform efforts to optimize cleaning, and potentially increasethe number of re-uses.

While it has herein been demonstrated that cleaning does not affect theion channel pharmacology of GABA_(A)Rs, it was not exhaustively testedthat the method in cells expressing other proteins. While severalstudies have characterized the effects of extracellularly applieddetergents on various receptors, the intracellular effect of LAS insmall concentrations is not well-understood. Thus to verify generalapplicability, the method should be validated using more channels,receptors, and cell types.

For many patch-clamp experiments, pipette internal solution containsingredients that degrade over time if not refrigerated (e.g. Adenosine5′-triphosphate magnesium salt, Guanosine 5′-triphosphate sodium salthydrate, phosphocreatine); thus, it is expected that pipettes aftermultiple cleanings will have reduced concentrations of theseingredients. It is hypothesized that maintaining a chilled (i.e., 4° C.)environment for pipette and internal solution, or devising a method forreplacing internal solution after every trial could mitigate thisdeleterious effect.

The cleaning process is typically faster than manually swapping pipettes(i.e., one minute versus approximately two minutes) and does not dependon operator experience, dexterity, or fatigue level. It requires nocomplex hardware additions to existing electrophysiology setups and noexpensive or caustic reagents. Therefore, it can be readily integratedinto different experimental preparations and coupled with existingtechniques that complement patch-clamp such as extracellularstimulation, two-photon microscopy, and optogenetics.

Pipette cleaning also facilitates experiments requiring specializedpipette tips. Various tip polishing techniques (using heat, pressure, orfocused ion beam) and coatings can improve the ability to obtainrecordings and their quality; however, these techniques are typicallytoo time-consuming to be routine. The ability to re-use thesecustom-shaped pipettes could make these involved techniques morepractical and scalable, reducing the need for automated pipettefabrication and inspection.

Further, the need for trained users to manually exchange pipettes isstill a barrier to large-scale patch-clamp studies in the brain. Increating the patcherBot, the first robot to successfully patch-clampmultiple cells without human intervention was demonstrated. In additionto automation, cleaning could also greatly facilitate simultaneousmulti-patch (i.e. dual, quadruple, octuple, etc.) experiments that havebeen instrumental in elucidating single-cell connectivity patterns inthe brain. Since cleaning is faster than manual pipette exchange andrequires no human intervention, it could increase the number andduration of simultaneous recordings.

Numerous characteristics and advantages have been set forth in theforegoing description, together with details of structure and function.While the invention has been disclosed in several forms, it will beapparent to those skilled in the art that many modifications, additions,and deletions, especially in matters of shape, size, and arrangement ofparts, can be made therein without departing from the spirit and scopeof the invention and its equivalents as set forth in the followingclaims. Therefore, other modifications or embodiments as may besuggested by the teachings herein are particularly reserved as they fallwithin the breadth and scope of the claims here appended.

What is claimed is:
 1. A control system comprising: a manipulatorassembly configured to manipulate an apertured surface to and from asample area and a cleaning area; and a controller configured to controlthe manipulator assembly; wherein the controller comprises a samplemodule configured to control the apertured surface through the samplearea, which is configured to contain biological sample targets; andwherein the control system enables re-use of the apertured surface withat least two different biological sample targets.
 2. The control systemof claim 1, wherein the controller further comprises: a cleaning moduleconfigured to control the apertured surface through the cleaning areaconfigured to contain cleaning solution, the cleaning area fluidicallyseparate from the sample area; and a testing module configured tocontrol the apertured surface into contact with a biological sampletarget of the biological sample targets to test for at least onecharacteristic of the biological sample target.
 3. The control system ofclaim 1, wherein the cleaning module is configured to control anautomated pressure assembly in communication with the apertured surface;wherein the cleaning module is further configured to control theapplication, by the automated pressure assembly, from negative topositive pressures relative to atmosphere to draw toward and expel fromthe apertured surface at least a portion of cleaning solution in thecleaning area; and wherein the cleaning module is further configured tocontrol the amount of cleaning solution drawn into the apertured surfaceso it reaches a height of a testing implement having the aperturedsurface, the height determined to sufficiently clean remnants of thetested sample target located at and beyond the apertured surface.
 4. Abiological sample testing system comprising: the control system of claim1; the apertured surface; and a re-use assembly comprising: the samplearea; and the cleaning area configured to contain cleaning solution, thecleaning area fluidically separate from the sample area; wherein thecontroller is further configured to: manipulate the apertured surfaceinto contact with a biological sample target of the biological sampletargets to test for at least one characteristic of the biological sampletarget; and manipulate the apertured surface to the cleaning area,wherein remnants of the tested biological sample target are cleaned fromthe apertured surface.
 5. A biological sample testing system comprising:a testing implement with a testing tip having an aperture; a manipulatorassembly configured to manipulate the testing implement; and a re-useassembly comprising: a sample area configured to contain biologicalsample targets; a cleaning area configured to contain cleaning solution,the cleaning area fluidically separate from the sample area; wherein themanipulator assembly is configured to: manipulate the aperture of thetesting implement into contact with a biological sample target of thebiological sample targets to test for at least one characteristic of thebiological sample target; and manipulate the testing implement to thecleaning area, wherein remnants of the tested biological sample targetare cleaned from the testing implement; wherein the re-use assemblyenables re-use of the testing tip of the testing implement with at leasttwo different biological sample targets.
 6. The biological sampletesting system of claim 5, wherein at least one characteristic of thetested biological sample target is selected from the group consisting ofan electrical activity, a molecular activity, a drug screening property,biological sample target type, a biophysical property, a morphologicalproperty and a genetic property.
 7. The biological sample testing systemof claim 5 further comprising a controller configured to control themanipulator assembly and the re-use assembly.
 8. The biological sampletesting system of claim 5, wherein the manipulator assembly isconfigured to manipulate the testing implement to the cleaning areawhether or not the test for at least one characteristic of thebiological sample target is successful.
 9. The biological sample testingsystem of claim 5 further comprising an automated pressure assembly incommunication with the testing tip of the testing implement; wherein theautomated pressure assembly is configured to apply from negative topositive pressures relative to atmosphere to draw toward and expel fromthe aperture at least a portion of cleaning solution in the cleaningarea; and wherein cleaning solution is drawn a height into the testingimplement sufficient to clean remnants of the tested sample targetlocated at and beyond the aperture.
 10. The biological sample testingsystem of claim 5 further comprising at least a second testing implementwith a testing tip having an aperture.
 11. The biological sample testingsystem of claim 5, wherein the sample area comprises two or morediscrete sample units fluidically separate from one another.
 12. Thebiological sample testing system of claim 5, wherein the cleaning areacomprises two or more discrete cleaning units fluidically separate fromone another.
 13. The biological sample testing system of claim 7,wherein the controller is a software control module.
 14. The biologicalsample testing system of claim 7, wherein the manipulator assemblycomprises an automated micro- or nano-positioner; and wherein thecontroller is configured to automatically control: manipulation of theaperture of the testing implement into contact with a first biologicalsample target of the biological sample targets; after the test,manipulation of the testing implement to the cleaning area; aftercleaning, manipulation of the testing implement to the sample area;manipulation of the aperture of the testing implement into contact witha second biological sample target of the biological sample targets, thesecond biological sample target different than the first biologicalsample target; and after the test, manipulation of the testing implementto the cleaning area.
 15. The biological sample testing system of claim13, wherein the controller further comprises hardware embedded with thesoftware control module.
 16. The biological sample testing system ofclaim 14, wherein the test of the first biological sample target and thetest of the second biological sample target are separately selected fromthe group consisting of successfully testing at least one characteristicand unsuccessfully testing at least one characteristic.
 17. A method ofbiological sample testing comprising: manipulating an apertured surfaceto and from a sample area and a cleaning area; controlling the aperturedsurface through the sample area, which is configured to containbiological sample targets; and re-using the apertured surface with atleast two different biological sample targets.
 18. The method of claim17, wherein controlling the apertured surface through the sample areacomprises: contacting the apertured surface with a first biologicalsample target of the biological sample targets; and testing for at leastone characteristic of the first biological sample target; and whereinre-using the apertured surface comprises: cleaning remnants of thetested biological sample target from the apertured surface; andcontacting the apertured surface with a second biological sample targetof the biological sample targets; wherein at least one characteristic ofthe tested biological sample targets is selected from the groupconsisting of an electrical activity, a molecular activity, a drugscreening property, biological sample target type, a biophysicalproperty, a morphological property and a genetic property.
 19. Themethod of claim 18, cleaning remnants of the tested biological sampletarget from the apertured surface comprises: applying from negative topositive pressures relative to atmosphere to draw toward and expel fromthe apertured surface at least a portion of cleaning solution in thecleaning area; and drawing a volume of cleaning solution into aperturedsurface sufficient to clean remnants of the tested sample target locatedat and beyond the apertured surface.
 20. The method of claim 19 furthercomprising automating the steps of the method via one or more outputsignals from a microprocessor executing commands in response to one ormore input signals; wherein the microprocessor executes instructionsstored in a memory that, when executed by the microprocessor, controlthe microprocessor to execute the commands.